Single-Cell RNA-Seq of Cultured Melanoma Cells

Cell suspensions was generated by washing melanoma cells from different cultures with PBS followed by trypsinizing with TripLE Xpress (Invitrogen, Darmstadt, Germany) for 5 minutes. The reaction was stopped by adding media containing FCS. Single melanoma cells from short-term cultures were captured on an integrated fluidic circuit RNA-seq chip (Fluidigm, Hamburg, Germany) using the Fluidigm C1 system. Cells were loaded onto the chip at a concentration of 200 cells/μL and imaged by phase-contrast. Cell capture, cell lysis, reverse transcription, and cDNA amplification were performed on the chip as described [50 (link)]. Illumina libraries were constructed using the Illumina Nextera XT DNA Sample Preparation kit using the protocol supplied by Fluidigm. Sequencing libraries were pooled (3 μL each) and purified with 18% SPRI beads. Library concentration and size distribution were assessed on an Agilent Bioanalyzer and with Qubit dsDNA HS Assay kits and a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Invitrogen, Darmstadt). Each cell was paired-end sequenced (100 base reads) on an Illumina HiSeq 2500 to a depth of 2–5 million reads and base-calling, adaptor trimming, and de-multiplexing were performed as described [51 (link), 52 (link)].